Not applicable
The present invention relates to improved methods for preparing support-bound nucleic acid arrays. More particularly, the invention relates to methods of preparing the arrays using dilute solutions of iodine to oxidize phosphite ester linkages to phosphate ester linkages.
Substrate-bound nucleic acid arrays, such as the Affymetrix DNA Chip, enable one to test hybridization of a target nucleic acid molecule to many thousands of differently sequenced nucleic acid probes at feature densities greater than about five hundred per 1 cm2. Because hybridization between two nucleic acids is a function of their sequences, analysis of the pattern of hybridization provides information about the sequence of the target molecule. The technology is useful for de novo sequencing and re-sequencing of nucleic acid molecules and also has important diagnostic uses in discriminating genetic variants that may differ in sequence by one or a few nucleotides. For example, substrate-bound nucleic acid arrays are useful for identifying genetic variants of infectious diseases, such as HIV, or genetic diseases, such as cystic fibrosis.
In one version of the substrate-bound nucleic acid array, the target nucleic acid is labeled with a detectable marker, such as a fluorescent molecule. Hybridization between a target and a probe is determined by detecting the fluorescent signal at the various locations on the substrate. The amount of signal is a function of the thermal stability of the hybrids. The thermal stability is, in turn, a function of the sequences of the target-probe pair: AT-rich regions of DNA melt at lower temperatures than GC-rich regions of DNA. This differential in thermal stabilities is the primary determinant of the breadth of DNA melting transitions, even for nucleic acids.
Depending upon the length of the nucleic acid probes, the number of different probes on a substrate, the length of the target nucleic acid, and the degree of hybridization between sequences containing mismatches, among other things, a hybridization assay carried out on a substrate-bound nucleic acid array can generate thousands of data points of different signal strengths that reflect the sequences of the probes to which the target nucleic acid hybridized. This information can require a computer for efficient analysis. The fact of differential fluorescent signal due to differences in thermal stability of hybrids complicates the analysis of hybridization results, especially from combinatorial nucleic acid arrays for de novo sequencing and custom nucleic acid arrays for specific re-sequencing applications. Modifications in custom array designs have contributed to simplifying this problem.
Further complications can arise and lead to variability in diagnostic or sequencing results. For example, degradation of nucleic acid probes, either during the synthesis steps or on standing can lead to variability in assay results. Accordingly, there exists a need for additional methods of nucleic acid array preparation, and the arrays themselves, to provide more robust tools for the skilled researcher. The present invention provides such methods and arrays.
In one aspect, the present invention provides methods for preparing nucleic acid arrays on a support. In these methods a plurality of nucleic acids are synthesized on the support and the synthesis steps include oxidizing a phosphite triester nucleic acid linkage to a phosphate triester nucleic acid linkage using a solution about 0.005 to about 0.05 M iodine in a mixture of water and organic solvent.
In one group of embodiments, each nucleic acid occupies a separate known region of the support, the synthesizing comprising:
(a) activating a region of the support;
(b) attaching a nucleotide to a first region, the nucleotide having a masked reactive site linked to a protecting group;
(c) repeating steps (a) and (b) on other regions of the support whereby each of the other regions has bound thereto another nucleotide comprising a masked reactive site linked to a protecting group, wherein the other nucleotide may be the same or different from that used in step (b);
(d) removing the protecting group from one of the nucleotides bound to one of the regions of the support to provide a region bearing a nucleotide having an unmasked reactive site;
(e) binding an additional nucleotide to the nucleotide with an unmasked reactive site;
(f) repeating steps (d) and (e) on regions of the support until a desired plurality of nucleic acids is synthesized, each nucleic acid occupying separate known regions of the support;
wherein the attaching and binding steps each form a phosphite triester linkage between the nucleotides and the unmasked reactive sites and further comprise oxidizing the phosphite triester linkage to a phosphate triester linkage with a solution of about 0.005 M to about 0.05 M iodine in an aqueous solvent mixture.
In another group of embodiments, the preparing comprises the sequential steps of.
a) removing a photoremoveable protecting group from at least a first area of a surface of a substrate, the substrate comprising immobilized nucleotides on the J2 surface, and the nucleotides capped with a photoremoveable protective group, without removing a photoremoveable protecting group from at least a second area of the surface;
b) simultaneously contacting the first area and the second area of the surface with a first nucleotide to couple the first nucleotide to the immobilized nucleotides in the first area, and not in the second area, the first nucleotide capped with a photoremoveable protective group;
c) removing a photoremoveable protecting group from at least a part of the first area of the surface and at least a part of the second area;
d) simultaneously contacting the first area and the second area of the surface with a second nucleotide to couple the second nucleotide to the immobilized nucleotides in at least a part of the first area and at least a part of the second area;
e) performing additional removing and nucleotide contacting and coupling steps so that a matrix array of at least 100 nucleic acids having different sequences is formed on the support;
with the proviso that the coupling steps further comprise oxidizing an initially formed phosphite ester to a phosphate ester using an aqueous solution of about 0.005 M to about 0.05 M iodine in an aqueous solvent mixture.
In another group of embodiments, the nucleoside phosphoramidite monomers used in the invention have the formula: 
wherein B represents adenine, guanine, thymine, cytosine, uracil or analogs thereof; R is hydrogen, hydroxy, protected hydroxy, halogen or alkoxy; P is a phosphoramidite group; and PG is a photoremoveable protected group.